We are continuing to make progress in our goal of defining germline modifiers of tumor progression and metastasis in PC. We have made extensive use of the well characterized C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) and FVB-Tg(ARR2/Pbsn-MYC)7Key/Nci (Hi-Myc) mouse models of prostate tumorigenesis to investigate the role of hereditary factors in the development of aggressive PC. The TRAMP mouse model develops neuroendocrine prostate cancer (NEPC), where tumorigenesis is induced through prostate-specific expression of the small and large SV40-T antigens that act to inactivate Rb and p53, which. At the time of diagnosis in humans NEPC is rare, accounting for <1% of prostate tumors. However, at the time of death, however, 25% of tumors are treatment-associated NEPCs that have arisen from a pre-existing adenocarcinoma after second- or third-line therapy. Our earlier work demonstrated that introducing hereditary variation by breeding into the TRAMP mouse substantially modulates disease aggressiveness since tumorigenesis is initiated by the same somatic event (i.e., expression of the SV40-T transgene) in each mouse. In the last year, we have used a QTL mapping approach to identify novel genes driving susceptibility to aggressive disease development by breeding TRAMP mice to Diversity Outbred (J:DO). J:DO mice are outbred stock derived from five classical laboratory strains (C57BL/6J, A/J, 129S1/SvImJ, NOD/ShiLtJ, and NZO/HlLtJ) and three wild-derived lines (CAST/EiJ, PWK/PhJ, and WSB/EiJ). They are maintained by random mating and are superior to other mapping populations in that they carry over 40 million SNPs, have fine recombination block structure and a high average minor allele frequency (i.e., 1/8th). Modifier locus mapping in a cohort of 493 (TRAMP x J:DO) F1 males revealed one locus spanning 3.3Mb on Chromosome 8 associated with distant metastasis (LOD=8.42, P=5.3x10-6). Eleven candidate genes within this locus were identified by integrating trait-correlation and expression quantitative trait locus data derived from RNA-seq analysis of 195 (TRAMP x J:DO) F1 tumors. The relevance of these eleven genes to aggressive human PC was investigated via in silico validation, which included three independent PC gene expression datasets and two PC genome-wide association studies (GWAS), encompassing data from over 5,300 PC patients. Three genes (RWDD4, CENPU, and CASP3) harbored variants associated with aggressive PC in GWAS analyses, and had expression levels associated with survival in all PC gene expression datasets. Dysregulation of RWDD4 and CENPU increased in vivo and in vitro aggressiveness of two human PC cell lines. Transcriptomic analysis of these cell lines revealed dysregulation of multiple tumor progression-associated pathways. This study demonstrates the utility of systems genetics as a means to define novel hereditary modifiers of aggressive PC. Further functional analysis of these genes is ongoing. In addition to this, in the last year we have completed a second study using a similar approach. Here, candidate metastasis susceptibility genes were identified through quantitative trait locus (QTL) mapping in 201 (TRAMPxPWK/PhJ) F2 males. Two metastasis-associated QTLs were identified; one on chromosome 12 (LOD = 5.86), and one on chromosome 14 (LOD = 4.41). Correlation analysis using microarray data from (TRAMPxPWK/PhJ) F2 prostate tumors identified 35 metastasis-associated transcripts within the two loci. The role of these genes in susceptibility to aggressive human PC was determined through in silico analysis using multiple datasets. First, analysis of candidate gene expression in two human PC datasets demonstrated that five candidate genes were associated with an increased risk of aggressive disease and lower disease-free survival. Second, four of these genes (GNL3, MAT1A, SKA3, and ZMYM5) harbored SNPs associated with aggressive tumorigenesis in the PLCO/CGEMS GWAS of 1,172 PC patients. Finally, over-expression of GNL3 and SKA3 in the PC-3 human PC cell line decreased in vitro cell migration and invasion. Functional characterization of these genes to clarify their role in metastasis is also ongoing. Finally, we are utilizing the Hi-Myc mouse model of prostate tumorigenesis, which represents a much more indolent form of prostate adenocarcinoma. Specifically, we are investigating how genetic variation enhances the propensity for aggressive PC development by crossing Hi-Myc females with DO males. Thus far, we have aged and phenotype a cohort of 276 transgene-positive (Hi-Myc x DO) F1 males, and have germline genotype data for 196 these animals. Significantly, we have observed pulmonary metastasis in >12% of these animals, which is of significance since metastasis has never been described in this mouse model of PC. QTL mapping studies are ongoing and aim to identify the modifier genes that increase susceptibility to metastasis in the Hi-Myc mouse model, and to assess their relevance to aggressive human PC.